Lung surfactant is critical in reducing surface tension and maintaining normal functions of lungs. Secretion of lung surfactant occurs via exocytosis of lamellar bodies in alveolar epithelial cells and is one of the most important aspects in lung surfactant homeostasis. However, the distal steps of the secretory process have not been studied extensively in type II cells. Our previous studies demonstrated an essential role of annexin II and SNAREs in the fusion of lamellar bodies with the plasma membrane. The broad, long-term objective of this proposal is to elucidate the molecular mechanisms of lung surfactant secretion from alveolar epithelial type II cells. Specific Aim I will test the hypothesis that SNAREs are organized in lipid rafts via their interaction with cholesterol and/or flotillins, and annexin II induces raft clustering to create the docking and fusion sites for exocytosis. Specific Aim II will examine the interaction of annexin II and SNAREs in greater detail, including the domain requirement for this interaction. SNAREs will be reconstituted into liposomes to test whether annexin II and SNAREs are sufficient for membrane fusion. The post-translational regulation of the annexin II-SNARE interaction will also be determined. Specific Aim III is to provide a link between our understanding of annexin II at the cellular level and its role at the animal level. The expression of annexin II in the type II cells of primary cultures and rat lungs will be knocked-down using SP-C promoter-driven small interfering RNA viral vectors. The physiological consequences in the annexin II knocked-down type II cells and animals will be examined. Deficiency of lung surfactant is the cause of respiratory distress syndrome in premature infants. Accomplishing the goals of this proposal will give a valuable direction to the therapy of pulmonary diseases such as neonatal respiratory distress syndrome.